Interfacial enzymes are a class of enzymes that comprise two domains in their proteinous structure; the first is a hydrophilic domain, while the second is a hydrophobic domain. This unique feature imparts this class of enzymes to favor the interfacial area once present in a two-phase system. Under these conditions, the active conformation is formed where the hydrophilic domain of the enzyme molecules faces the aqueous layer while the hydrophobic domain faces the hydrophobic layer.
Lipases and phospholipases are the most familiar interfacial enzymes that express their catalytic activity once present in an interfacial system. Lipases (triacylglycerol hydrolase E.C. 3.1.1.3) are defined as hydrolytic enzymes that act on the ester linkage in triacylglycerol in aqueous systems to yield free fatty acids, partial glycerides and glycerol. Phospholipases also belong to the class of hydrolytic enzymes, however they cleave favorably and specifically the ester linkage of phospholipids present in aqueous systems, to yield free fatty acids, lysophospholipids, glycerophospholipids, phosphatidic acid and free alcohol, depending on the type of phospholipase.
Lipases and phospholipases are widely distributed among animals, plants and microorganisms. The interest in the industrial application of lipases and phospholipases has been rapidly growing during the last two decades. It has been found that under low water activity this class of enzymes catalyzes their reverse hydrolysis reaction. The reverse catalytic activity of lipases and phospholipases has been widely exploited for the synthesis of valuable compounds that contain ester and amide linkages or other related chemicals containing functional groups such as hydroxyl, carboxylic and amino groups. In particular, lipases and phospholipases have been utilized for reforming fats, oils, waxes, phospholipids and sphingolipids to obtain new desired functional properties, and for separating optically active compounds from their racemic mixtures. Of particular interest is the use of interfacial enzymes for the synthesis of short-chain alkyl esters (biodiesel), disclosed herein.
Currently, there are more than 40 different lipases and phospholipases commercially available, however only a few of them are prepared in commercial quantities. Some of the most industrially promising interfacial enzymes are derived from Candida antarctica, Candida rugosa, Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camernbertii, Alcaligenes sp., Burhholderia sp., Thermomyces lanuginosa, Chromobacterium viscosum, papaya seeds, and pancreatin.
Immobilization of enzymes has been described by a vast number of techniques basically aiming at reducing the cost contribution of enzymes in the overall process, facilitating the recovery of enzymes from the products and enabling continuous operation of the process. Immobilization techniques are in general divided according to the following:                1. Physical adsorption of enzymes to solid supports, such as silica and insoluble polymers.        2. Adsorption on ion-exchange resins.        3. Covalent binding of enzymes to a solid support material, such as epoxidated inorganic or polymer supports.        4. Entrapment of enzymes in a growing polymer.        5. Confinement of enzymes in a membrane reactor or in semi-permeable gels.        6. Cross-linking enzyme crystals (CLECS's) or aggregates (CLEAS's).        
All the aforementioned enzyme immobilization procedures are comprised of the following steps:                1. Dissolving the enzyme in an appropriate buffer system with respect to pH, temperature, type of buffer salts and ionic strength.        2. Adding the solid support into the enzyme solution and mixing for some time until enzyme molecules are immobilized on the solid support.        3. Filtering off the solid support which contains the immobilized enzyme.        4. Washing the support with an appropriate buffer to remove loosely bound enzyme molecules and then drying the solid support.        
Interfacial enzymes, mostly lipases, have been immobilized following the aforementioned techniques. These offered immobilized enzyme preparations possessing low synthetic activity and/or short operational half-life time. In an attempt to increase the synthetic activity of immobilized lipases and other interfacial enzymes different activation methods have been applied. These methods include:                1. Binding the surface functional groups of enzymes with hydrophobic residues such as fatty acids or polyethylene glycol.        2. Coating the surface of enzymes with surfactants, such as polyol fatty acid esters.        3. Contacting enzymes with hydrophobic supports, typically polypropylene, which have been pretreated with hydrophilic solvents, such as ethanol or iso-propanol.        4. Adding enzyme activators, such as salt solution, glycerol, etc. at low concentration, typically below 1%, into the reaction system.        
None of the above mentioned methods yielded satisfactory results with respect to activation, stabilization and cost-effectiveness of immobilized interfacial enzymes in order to carry out enzymatic reverse conversions at industrial quantities. Also, it has been reported that most enzymes, when immobilized according to the aforementioned procedure, either lose a significant portion of their synthetic activity or do not exhibit their full activity performance due to certain constraints imposed by the immobilization procedure. For example, coating lipases and phospholipases with polyol fatty acid esters encountered a serious challenge where lipase molecules were not fully coated with the activator; therefore those enzyme molecules not brought into contact with the activator, remained inactive.
Another major drawback of lipases and phospholipases is their low tolerance towards hydrophilic substrates, particularly short-chain alcohols and short-chain fatty acids (below C4). It has been observed in many research studies that short-chain alcohols and short-chain fatty acids, such as methanol and acetic acid, are responsible for detaching essential water molecules from the quaternary structure of those enzymes, leading to their denaturation and consequently loss of their catalytic activity. This drawback has prohibited the application of lipases for production of commercial quantities of fatty acid methyl esters “biodiesel” using oil triglycerides and methanol as substrates.
It is therefore an object of this invention to provide a new method for obtaining highly active and stable immobilized, interfacial enzymes, in particular lipases and phospholipases for synthetic applications. Of particular interest is the use of these enzymes for the synthesis of fatty acid short-chain alkyl esters for use as “biodiesel”.
It is a further object of the present invention to provide highly active, stable, immobilized interfacial enzymes, possessing high tolerance towards short-chain alcohols, such as methanol, ethanol and glycerol, and short-chain fatty acids, such as acetic acid.
These and other objects of the invention will become apparent as the description proceeds.